US20110015133A1 - Pax2 and pax8 as tumour targets for immunologic and molecular treatment strategies - Google Patents

Pax2 and pax8 as tumour targets for immunologic and molecular treatment strategies Download PDF

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US20110015133A1
US20110015133A1 US12/377,647 US37764707A US2011015133A1 US 20110015133 A1 US20110015133 A1 US 20110015133A1 US 37764707 A US37764707 A US 37764707A US 2011015133 A1 US2011015133 A1 US 2011015133A1
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pax2
cell
pax8
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Ulrich Keilholz
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Charite Universitaetsmedizin Berlin
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001152—Transcription factors, e.g. SOX or c-MYC
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6875—Nucleoproteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6878—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids in eptitope analysis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55522—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6081—Albumin; Keyhole limpet haemocyanin [KLH]

Definitions

  • the present invention refers to the transcription factors PAX2 and PAX8 expressed in solid tumours and haematologic malignancies, and their utility as a target in immunotherapy and molecular therapy.
  • the invention refers to a method for identifying an immunogenic T-cell epitope from PAX2 and/or PAX8.
  • the invention refers to a use of immunogenic T-cell epitopes, e.g. identified by said method, and their use as targets for the recognition by targeting means, e.g. T-cells or antibodies.
  • the invention also refers to peptides representing immunogenic T-cell epitopes and their uses for the preparation of a pharmaceutical composition for immunotherapy of PAX2 and/or PAX8 expressing malignancies.
  • the transcription factor WT1 is an antigen, which has recently attracted much interest as target for cancer immunotherapy (1).
  • WT1 was connected with Wilms' tumour, an embryonal malignancy of the kidney affecting infants and young children.
  • WT1 is highly expressed in leukaemic blasts and is also broadly expressed in solid tumours.
  • Recent papers show its wide expression in various solid tumour samples, including common malignancies as colorectal cancer, breast cancer and lung cancer.
  • WT1 plays a key pathogenic role as down-regulation of the wt1 gene leads to growth arrest, differentiation and apoptosis.
  • WT1 peptides in patients with acute leukaemia showing good immunogenicity and clinical efficacy of the vaccine (3,4,5).
  • PAX2 and PAX8 are closely related transcription factors, are expressed in WT and are potentially involved in its induction.
  • PAX paired axial
  • PAX paired axial
  • the PAX (paired axial) genes belong to the hombeobox gene family of transcription factors. These proteins have sequence homology to the Drosophila segmentation genes paired and gooseberry.
  • the mammalian PAX proteins are numbered 1-9, all containing the so-called “paired box” sequence, a 124 amino-acid conserved domain.
  • PAX2 and PAX proteins are closely related Both PAX2 and PAX8 genes play a role in the regulation of WT1 expression in the developing kidney.
  • PAX2 and PAX8 are also expressed in various types of cancer including lymphomas, which do not express WT1.
  • AML human acute myeloid leukaemia
  • PAX8 is a cause of up-regulation of WT1 in human acute myeloid leukaemia (AML), breast and colorectal carcinomas (9,10). Therefore, in addition to WT1 as target for immunotherapy itself, it is of great interest to exploit the WT1 expression regulators PAX2 and PAX8 as cancer vaccination antigen.
  • the patent U.S. Pat. No. 6,723,506 discloses the finding of a fusion oncogene designated PAX8-PPAR ⁇ 1 in carcinoma samples.
  • the fusion oncogene (or its reciprocal PPAR ⁇ 1-PAX8) is the result of a chromosomal translocation fusing chromosomes 2 and 2.
  • Molecular characterization of PAX8-PPAR ⁇ 1 revealed nucleotide and amino acid sequences useful for detection and treatment of certain tumours, particularly thyroid follicular carcinomas.
  • the patent U.S. Pat. No. 6,071,697 provides a method for testing the differentiation status of pancreatic cells in mammals using two members of the PAX family, namely PAX4 and PAX6.
  • a deficiency in PAX4 expression is indicative of deficiency or failure in ⁇ -cell development and thus insulin production. Accordingly, the method is useful for determining the risk of developing juvenile diabetes.
  • Further disclosed are a method for testing a medicament for a gene therapy approach and transgenic mammals comprising an inactivated PAX4 allele and optionally and inactivated PAX6 allele.
  • TAAs tumour-associated antigens
  • a prerequisite for successful immunotherapy is the knowledge of immunogenic T-cell epitopes.
  • the search for new T-cell epitopes in known TAAs using the classical “reverse immunology” strategy has led to the identification of several T-cell epitopes, most of them restricted to HLA-A2 (also referred to as HLA-A*02) (11).
  • HLA-A*02 also referred to as HLA-A*02
  • This strategy includes the prediction of potential T-cell epitopes from known tumour antigens, their analysis for MHC-binding, followed by the in vitro generation of peptide-reactive T-cells and their testing of target cell recognition.
  • T-cell epitopes A more recent approach to increase the efficacy of identifying potential T-cell epitopes is the ex vivo screening of candidate epitopes for recognition by T-cells from patients which are naturally primed against the target antigen (12-15).
  • Candidate epitopes can be predicted using appropriate prediction models in silico (e.g. ref. 16).
  • WO 00/18795 used the BIMAS HLA peptide binding prediction analysis (17).
  • An elegant approach to show epitope processing is by in vitro proteasome-mediated digestion pattern analysis. This approach has reliably identified HLA-A2 binding epitopes from tumour and viral proteins using 25-30-mer peptides encompassing the putative epitope (18,19). Even though such in silico prediction models as mentioned above may be highly efficacious to predict candidate epitopes, the necessity remains to verify their utility in vitro and in vivo.
  • An object of the present invention is to provide immunogenic T-cell epitopes useful in immunotherapy of malignancies.
  • the object of the present invention is solved by an immunogenic T-cell epitope represented by a peptide selected from the group consisting of peptides according to SEQ ID No. 5 (3496), SEQ ID No. 6 (3497), SEQ ID No. 16 (3520), SEQ ID No. 20 (3519), SEQ ID No. 21 (3518), SEQ ID No. 29 (3550), and SEQ ID No. 32 (3553).
  • the object of the present invention is also solved by an immunogenic T-cell epitope represented by a peptide according to SEQ ID No. 41 (277-285) or SEQ ID No. 42 (323-333).
  • the immunogenic T-cell epitope is from PAX2 and/or PAX8, preferably from PAX2.
  • the object of the present invention is further solved by a use of an immunogenic T-cell epitope from PAX2 and/or PAX8, preferably from PAX2, as a target for recognition by a targeting means.
  • the T-cell epitope is a HLA binding T-cell epitope, preferably selected from a HLA-A2, HLA-A1 or HLA-A24 binding T-cell epitope.
  • the T-cell epitope is represented by a peptide selected from the group of peptides according to SEQ ID No. 5 (3496), SEQ ID No. 6 (3497), SEQ ID No. 16 (3520), SEQ ID No. 20 (3519), SEQ ID No. 21 (3518), SEQ ID No. 29 (3550), and SEQ ID No. 32 (3553).
  • the T-cell epitope is represented by a peptide according to SEQ ID No. 41 (277-285) or SEQ ID No. 42 (323-333).
  • the T-cell epitope is represented by the peptide according to SEQ ID No. 5 (3496).
  • three T-cell epitopes are used represented by the peptides according to SEQ ID No. 16 (3520), SEQ ID. No 29 (3550), and SEQ ID No. 32 (3553).
  • two T-cell epitopes are used represented by the peptides according to SEQ ID No. 5 (3496) and SEQ ID No. 6 (3497).
  • two T-cell epitopes are used represented by the peptides according to SEQ ID No. 20 (3519) and SEQ ID No. 21 (3518).
  • the targeting means is a component of the immune system, preferably a T-cell or an antibody.
  • the antibody is selected from the group consisting of monoclonal antibodies, polyclonal antibodies, homobodies, epibodies, Fc fragments and portions or variants thereof.
  • the object of the present invention is further solved by a use of a peptide selected from the group consisting of peptides according to SEQ ID No. 5 (3496), SEQ ID No. 6 (3497), SEQ ID No. 16 (3520), SEQ ID No. 20 (3519), SEQ ID No. 21 (3518), SEQ ID No. 29 (3550), and SEQ ID No. 32 (3553) for the preparation of a pharmaceutical composition for the treatment of a PAX2 and/or PAX8 expressing disease, preferably a PAX2 expressing disease.
  • the object of the present invention is further solved by a use of a peptide according to SEQ ID No. 41 (277-285) or SEQ ID No. 42 (323-333) for the preparation of a pharmaceutical composition for the treatment of a PAX2 and/or PAX8 expressing disease,
  • the disease is selected from renal cancer, colorectal cancer, breast cancer, and ovarian cancer.
  • the peptide according to SEQ ID No. 5 (3496) is used for the preparation, and the disease preferably is renal cancer.
  • the peptide according to SEQ ID No. 41 (277-285) is used for the preparation, and the disease preferably is renal cancer.
  • the peptides according to SEQ ID No. 16 (3520), SEQ ID No. 29 (3550), and SEQ ID No. 32 (3553) are used for the preparation, and the disease preferably is colorectal cancer.
  • the peptides according to SEQ ID No. 5 (3496) and 3497 SEQ ID No. 6 (3497) are used for the preparation, and the disease preferably is colorectal cancer.
  • the peptides according to SEQ ID No. 20 (3519) and SEQ ID No. 21 (3518) are used for the preparation, and the disease preferably is colorectal cancer.
  • the peptide according to SEQ ID No. 42 (323-333) is used for the preparation, and the disease preferably is ovarian cancer.
  • the object of the present invention is further solved by a use of a peptide selected from the group consisting of peptides according to SEQ ID No. 5 (3496), SEQ ID No. 6 (3497), SEQ ID No. 16 (3520), SEQ ID No. 20 (3519), SEQ ID No. 21 (3518), SEQ ID No. 29 (3550), and SEQ ID No. 32 (3553) for the preparation of a pharmaceutical composition for raising an immune response in a subject, preferably a mammal.
  • the object of the present invention is further solved by a use of a peptide according to SEQ ID No. 41 (277-285) or SEQ ID No. 42 (323-333) for the preparation of a pharmaceutical composition for raising an immune response in a subject, preferably a mammal.
  • the peptide according to SEQ ID No. 5 (3496) is used for the preparation.
  • the peptides according to SEQ ID No. 16 (3520), SEQ ID No. 29 (3550), and SEQ ID No. 32 (3553) are used for the preparation.
  • the peptides according to SEQ ID No. 5 (3496) and 3497 SEQ ID No. 6 (3497) are used for the preparation.
  • the peptides according to SEQ ID No. 20 (3519) and SEQ ID No. 21 (3518) are used for the preparation.
  • the immune response is raised by contacting the pharmaceutical composition with T-cells of a subject, preferably a mammal, in vivo.
  • the immune response is raised by contacting the pharmaceutical composition with T-cells of a subject, preferably a mammal, ex vivo.
  • the object of the present invention is further solved by a pharmaceutical composition
  • a pharmaceutical composition comprising a peptide selected from the group consisting of peptides according to SEQ ID No. 5 (3496), SEQ ID No. 6 (3497), SEQ ID No. 16 (3520), SEQ ID No. 20 (3519), SEQ ID No. 21 (3518), SEQ ID No. 29 (3550), and SEQ ID No. 32 (3553).
  • the object of the present invention is further solved by a pharmaceutical composition
  • a pharmaceutical composition comprising a peptide according to SEQ ID No. 41 (277-285) or SEQ ID No. 42 (323-333).
  • the pharmaceutical composition comprises a peptide according to SEQ ID No. 5 (3496).
  • the pharmaceutical composition comprises three peptides according to SEQ ID No. 16 (3520), SEQ ID No. 29 (3550), and SEQ ID No. 32 (3553).
  • the pharmaceutical composition comprises two peptides according to SEQ ID No. 5 (3496) and 3497 SEQ ID No. 6 (3497).
  • the pharmaceutical composition comprises two peptides according to SEQ ID No. 20 (3519) and SEQ ID No. 21 (3518).
  • the object of the present invention is further solved by a use of a targeting means according to the present invention for the preparation of a pharmaceutical composition for the treatment of a PAX2 and/or PAX8-expressing disease, preferably a PAX2-expressing disease, most preferably selected from renal cancer, colorectal cancer, breast cancer, and ovarian cancer.
  • a targeting means for the preparation of a pharmaceutical composition for the treatment of a PAX2 and/or PAX8-expressing disease, preferably a PAX2-expressing disease, most preferably selected from renal cancer, colorectal cancer, breast cancer, and ovarian cancer.
  • the targeting means is a T-cell or an antibody directed against an immunogenic T-cell epitope from PAX2 and/or PAX8, preferably from PAX2.
  • the T-cell epitope is represented by a peptide selected from the group consisting of peptides according to SEQ ID No. 5 (3496), SEQ ID No. 6 (3497), SEQ ID No. 16 (3520), SEQ ID No. 20 (3519), SEQ ID No. 21 (3518), SEQ ID No. 29 (3550), and SEQ ID No. 32 (3553).
  • the T-cell epitope is represented by a peptide according to SEQ ID No. 41 (277-285) or SEQ ID No. 42 (323-333).
  • the antibody is selected from the group consisting of monoclonal antibodies, polyclonal antibodies, homobodies, epibodies, Fc fragments and portions or variants thereof.
  • the object of the present invention is further solved by a pharmaceutical composition comprising a targeting means according to the present invention.
  • the targeting means is a T-cell or an antibody directed against an immunogenic T-cell epitope from PAX2 and/or PAX8, preferably from PAX2.
  • the T-cell epitope is represented by a peptide selected from the group consisting of peptides according to SEQ ID No. 5 (3496), SEQ ID No. 6 (3497), SEQ ID No. 16 (3520), SEQ ID No. 20 (3519), SEQ ID No. 21 (3518), SEQ ID No. 29 (3550), and SEQ ID No. 32 (3553).
  • the T-cell epitope is represented by a peptide according to SEQ ID No. 41 (277-285) or SEQ ID No. 42 (323-333).
  • the targeting means is a T-cell or an antibody, directed against a peptide selected from the group consisting of peptides according to SEQ ID No. 5 (3496), SEQ ID No. 6 (3497), SEQ ID No. 16 (3520), SEQ ID No. 20 (3519), SEQ ID No. 21 (3518), SEQ ID No. 29 (3550), and SEQ ID No. 32 (3553).
  • the targeting means is a T-cell or an antibody, directed against a peptide according to SEQ ID No. 41 (277-285) or SEQ ID No. 42 (323-333).
  • the antibody is selected from the group consisting of monoclonal antibodies, polyclonal antibodies, homobodies, epibodies, Fc fragments and portions or variants thereof.
  • the object of the present invention is further solved by a method for identifying an immunogenic T-cell epitope, a portion or a variant thereof comprising the following steps:
  • the T-cell epitope is a HLA binding T-cell epitope, preferably selected from HLA-A2, HLA-A1 and HLA-A24 binding T-cell epitope.
  • the candidate epitope is excluded from the highly conserved paired box sequence of PAX2 and/or PAX8, preferably from PAX2.
  • the T-cells in step (b) are obtained from a tumour patient having a PAX2 and/or PAX8-expressing disease, preferably a PAX2-expressing disease, most preferably selected from renal cancer, colorectal cancer, breast cancer, and ovarian cancer.
  • the T-cell clones are useful for demonstrating natural epitope processing.
  • the object of the present invention is further solved by a use of the method according to the present invention for identifying an immunogenic T-cell epitope from PAX2 and/or PAX8, preferably from PAX2.
  • the object of the present invention is further solved by a use of the immunogenic T-cell epitope from PAX2 and/or PAX8, preferably from PAX2, identified by the method according to the present invention, as a target for a targeting means.
  • the object of the present invention is further solved by a use of an immunogenic T-cell epitope from PAX2 and/or PAX8, preferably from PAX2, identified by the method according to the present invention, for the preparation of a pharmaceutical composition for the treatment of a PAX2 and/or PAX8-expressing disease, preferably a PAX2-expressing disease.
  • the object of the present invention is further solved by a pharmaceutical composition
  • a pharmaceutical composition comprising an immunogenic T-cell epitope from PAX2 and/or PAX8, preferably from PAX2, identified by the method according to the present invention.
  • the wording “use of an immunogenic T-cell epitope ( . . . ) as a target for recognition by a targeting means” may have a dual meaning.
  • the T-cell epitope e.g. represented by a peptide
  • the exogenous T-cell epitope serves a an antigenic target for endogenous components of the immune system.
  • the T-cell epitope is part of endogenous PAX2 and/or PAX 8, and as such serves as a target for endogenous components of the immune system, e.g. T-cells and antibodies, which were raised previously in a subject in response to a contact with the administered T-cell epitope.
  • a pharmaceutical composition for raising an immune response comprises the meaning that the pharmaceutical composition is used as a vaccine for active immunisation.
  • a pharmaceutical composition comprising a targeting means, e.g. T-cells or an antibody generated in vitro, can be used as for passive immunisation.
  • contacting the pharmaceutical composition with T-cells ( . . . ) in vivo means that the pharmaceutical composition is administered to a subject, e.g. by sequential s.c. injections.
  • T-cell therapy comprises the meaning that T-cells are removed from a subject, contacted with the pharmaceutical composition in vitro, and finally infused into a subject (so-called T-cell therapy).
  • Donor and acceptor of the T-cells may be identical or may be different.
  • the donor and acceptor species may be identical or may be different.
  • the present invention refers to two transcription factors, PAX2 and PAX8 (paired box genes 2 and 8), expressed in various cancers and haematologic malignancies and their utility as a target in immunotherapy and molecular therapy.
  • PAX2 and PAX8 paired box genes 2 and 8
  • the invention refers to the PAX proteins excluding the highly conserved paired box sequence (PAX2 AA 16-141, PAX 8 9-216) comprising T-cell epitopes or a portion or variant thereof.
  • PAX2 or PAX8 is a cause of up-regulation of WT1 in human AML, breast and colorectal carcinomas (9,10), we set out to provide tools and their uses for cancer immunotherapy and other therapies targeting PAX2 and PAX8.
  • HLA-A2 binding candidate epitopes which are spontaneously immunogenic in colorectal cancer patients.
  • the unique sequence of the 416 amino acid PAX2 protein is located at amino acids 145 to 191.
  • the highly conserved portion of the paired box domain (amino acids 16 to 140) and the homeobox (amino acids 235 to 278) are excluded from epitope identification.
  • the paired box is located at amino acids 9 to 216, amino acids 228 to 250 form the homeobox homology region, and a further highly conserved octapeptide sequence that is excluded from epitope identification is located at amino acids 180 to 187.
  • T-cell epitopes An approach to efficiently identify potential T-cell epitopes is the ex vivo screening of candidate epitopes for recognition by T-cells from patients which are naturally primed against the target antigen (20-22). Using this approach we could identify HLA-A2 binding candidate epitopes from PAX2 against which patients with colorectal cancer have spontaneous T cell responses.
  • PBMC Peripheral blood mononuclear cells
  • HLA-A2 HLA-A*0201 binding candidate epitopes (nona- and decamers) from PAX2 predicted using the SYFPEITHI algorithm
  • PAProC prediction of Peptide No. Amino acid proteasomal digestion
  • SEQ ID No. Position sequence WT Prot Immuno Const Score 3469 295 ⁇ A L T P G L D E V I ⁇ ⁇ /+ 31
  • SEQ ID No. 1 3480 292 ⁇ S L P A L T P G L — +/ ⁇ +/ ⁇ 27
  • SEQ ID No. 2 3485 299 ⁇ G L D E V K S S L I; II ⁇ +/ ⁇ 25 SEQ ID No.
  • Peptides were synthesized in an Applied Biosystems 432A peptide synthesizer following standard protocols. Synthesized products were analysed by high-performance liquid chromatography (Varian Star; Zinsser Analytics, Kunststoff, Germany) and MALDI-TOF mass spectometry (G2025A; Hewlett-Packard, Waldbronn, Germany), and purified by preparative HPLC to purities >95%.
  • Antigen-specific T-cells were detected by a functional assay. Intracellular IFN ⁇ accumulation induced by WT1 peptide was assessed by flow cytometry as described previously (2).
  • PAX2 and PAX8 expression in colorectal cancer cell lines (ratio PAX/PBGD determined by quantitative RT-PCR) Cell line Expression PAX2 Expression PAX8 Colo 205 1.64E ⁇ 04 2.00E ⁇ 04 Colo 206F 3.98E ⁇ 04 3.76E ⁇ 05 Colo 320 1.60E ⁇ 05 3.29E ⁇ 05 Cx 2 1.68E ⁇ 04 1.88E ⁇ 05 Cx 94 3.93E ⁇ 03 2.22E ⁇ 05 DLD 1 1.00E ⁇ 08 3.18E ⁇ 05 HCT 116 2.62E ⁇ 02 7.00E ⁇ 05 HT 29 1.13E ⁇ 04 3.89E ⁇ 05 SW 403 8.96E ⁇ 06 1.64E ⁇ 04 SW 480 1.24E ⁇ 04 7.19E ⁇ 05 SW 620 2.69E ⁇ 04 4.01E ⁇ 05 SW 948 1.10E ⁇ 04 2.13E ⁇ 05 CaCo 2 2.24E ⁇ 05 2.49E ⁇ 06 LST 174 1.00E ⁇ 08 1.28E ⁇ 05
  • the protocol given in (1) describing the vaccination with WT1.126-134 in a HLA-A2 (HLA-A*0201) positive patient is applied.
  • An HLA-A2 positive patient diagnosed with PAX2 positive cancer receives eight biweekly vaccinations with the peptide in a dose of 0.2 mg admixed with 1 mg keyhole limpet hemocyanin (KLH, Immucothel, biosyn, Germany) as adjuvant i.d. and s.c. on day 0.
  • KLH keyhole limpet hemocyanin
  • GM-CSF Leukomax, Essex Pharma, Germany
  • the combination of both ajuvants is chosen due to the immunological efficacy in melanoma peptide vaccination (23). Vaccination cycles can be repeated if necessary.
  • T-cell response to PAX2 peptides is performed in a peripheral blood using tetramer and intracellular IFN ⁇ staining by flow cytometry (2).
  • T-cell epitopes To identify T-cell epitopes, they were screened for recognition by T-cells from patients with renal cell and ovarian cancer which are PAX8 positive tumours secreting cytokine IFN ⁇ or TNF ⁇ in response to PAX 8 or control HIV peptide. By using 4 pools of three peptides each, every peptide was present in two pools. Two epitopes, 277-285 and 323-333, were recognized in both pools, thus verifying them as epitopes (Table VI).

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JP6875126B2 (ja) * 2014-01-21 2021-05-19 アルバート アインシュタイン カレッジ オブ メディシン 迅速かつ包括的なｔ細胞免疫モニタリング用の細胞プラットフォーム
KR20190019068A (ko) 2016-05-18 2019-02-26 큐 바이오파마, 인크. T-세포 조절 다량체 폴리펩타이드 및 이의 사용 방법
EP3458095A4 (fr) 2016-05-18 2019-11-27 Albert Einstein College of Medicine Variants de polypeptides pd-l1, polypeptides multimères modulateurs des lymphocytes t et procédés d'utilisation correspondants
EP3558339B1 (fr) 2016-12-22 2024-01-24 Cue Biopharma, Inc. Polypeptides multimères modulateurs des lymphocytes t et leurs méthodes d'utilisation
EP3565829A4 (fr) 2017-01-09 2021-01-27 Cue Biopharma, Inc. Polypeptides multimères modulateurs de lymphocytes t et leurs procédés d'utilisation
IL269000B2 (en) 2017-03-15 2024-06-01 Cue Biopharma Inc Methods for modulating an immune response
WO2019139896A1 (fr) 2018-01-09 2019-07-18 Cue Biopharma, Inc. Polypeptides multimères modulateurs de lymphocytes t et leurs procédés d'utilisation
KR20230009872A (ko) 2020-05-12 2023-01-17 큐 바이오파마, 인크. 다량체 t-세포 조절 폴리펩타이드 및 이의 사용 방법

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